Locations and molecular forms of PACAP and sites and characteristics of PACAP receptors in canine ileum.
In canine ileum we investigated the distribution of pituitary adenylate cyclase-activating peptide (PACAP), using immunofluorescence and radioimmunoassay and the binding of 125I-PACAP-27 to membranes. Nerve profiles immunoreactive to PACAP-27, and often to vasoactive intestinal polypeptide (VIP) as well, were found in all plexi, but PACAP was present in approximately 100-fold lesser amounts than VIP. High-performance liquid chromatography analysis of deep muscular plexus (DMP) synaptosomes suggested the presence of PACAP-38, PACAP-27, and a third unidentified molecular form. High- and low-affinity 125I-PACAP-27 binding sites were found in DMP synaptosomes and circular smooth muscle (CM) plasma membranes. In competition studies with DMP membranes, high (H)- and low (L)-affinity dissociation constants (Kd) and maximal binding capacities (Bmax) were as follows: KdH = 66.9 pM, BmaxH = 101 fmol/mg; KdL = 2.18 nM, BmaxL = 580 fmol/mg protein. The binding of 125I-PACAP-27 was fast. Dissociation was slow and incomplete in the presence of unlabeled PACAP-27 but accelerated by pretreatment with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). GTP gamma S or cholera toxin treatment eliminated high-affinity binding in both membranes. VIP had approximately 100-fold lower affinity than PACAP-27 in both membranes. Cross-linking studies identified an approximately 70-kDa PACAP receptor in each membrane. Thus PACAP coexists with VIP in ileal enteric nerves and acts on PACAP-preferring, possibly Gs-coupled, receptors in DMP synaptosomes and CM membranes.
Mao
Y K
YK
Department of Biomedical Science, McMaster University, Hamilton, Ontario, Canada.
Wang
Y F
YF
Moogk
C
C
Fox-Threlkeld
J E
JE
Xiao
Q
Q
McDonald
T J
TJ
Daniel
E E
EE
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004285:Dogs; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007082:Ileum / Q000166:cytology / Q000294:innervation* / Q000378:metabolism*; D007700:Kinetics; D009130:Muscle, Smooth / Q000166:cytology / Q000294:innervation / Q000378:metabolism; D009197:Myenteric Plexus / Q000166:cytology / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009479:Neuropeptides / Q000032:analysis / Q000378:metabolism*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone / Q000302:isolation & purification / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism; D014660:Vasoactive Intestinal Peptide / Q000032:analysis
D003432:Cross-Linking Reagents; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone; D014660:Vasoactive Intestinal Peptide; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002772:Cholera Toxin